Journal
ANALYTICAL BIOCHEMISTRY
Volume 526, Issue -, Pages 43-49Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2017.03.010
Keywords
Phosphatidylcholine-specific phospholipase C; Diacylglycerol; Phosphatidylcholine; Liquid chromatography-mass spectrometry
Funding
- MEXT/JSPS KAKENHI [22370047, 15J05821, 23116505, 25116704, 26291017, 15K14470]
- Japan Science and Technology Agency [AS221Z00794F, AS231Z00139G, AS251Z01788Q, AS2621643Q]
- Naito Foundation
- Hamaguchi Foundation for the Advancement of Biochemistry
- Daiichi-Sankyo Foundation of Life Science
- Terumo Life Science Foundation
- Futaba Electronic Memorial Foundation
- Daiwa Securities Health Foundation
- Japan Foundation for Applied Enzymology
- Food Science Institute Foundation
- Skylark Food Science Institute
- Asahi Group Foundation
- Ono Medical Research Foundation
- Japan Milk Academic Alliance
- Grants-in-Aid for Scientific Research [26291017, 15J05821, 15K14470, 17H03650] Funding Source: KAKEN
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Phosphatidylcholine (PC)-specific phospholipase C (PC-PLC) hydrolyzes PC to generate the second messenger 1,2-diacylglycerol (DG) and phosphocholine. PC-PLC plays pivotal roles in inflammation, carcinogenesis, tumor progression, atherogenesis, and subarachnoid hemorrhage. Although the activity of PC-PLC in mammalian tissues was discovered approximately 40 years ago, neither the protein nor its gene has been identified. In the present study, we developed a non-radioactive enzyme activity assay for PC-PLC based on mass spectrometric detection of DG following HPLC separation. This new liquid chromatography-mass spectrometry (LC-MS) assay directly determines a specific reaction product, 1-palmitoyl-2-oleoyl-DG, that is generated from 1-palmitoyl-2-oleoyl-PC by purified Bacillus cereus PC-PLC. The LC-MS assay offers several advantages including a lower background (0.02% versus 91%), higher signal background ratio (4242 versus 1.06)/signal noise ratio (7494 versus 4.4), higher sensitivity (>= 32-fold), and lower limit of quantitation (0.04 pmol versus 0.69 pmol of PC-PLC), than a conventional fluorometric assay, which indirectly detects phosphocholine produced in the reaction. In addition to Bacillus cereus PC-PLC, the LC-MS assay was applicable to the measurement of mammalian PC-PLC prepared from the mouse brain. The radioisotope-free, highly sensitive and precise LC-MS assay for PC-PLC would be useful for the purification and identification of PC-PLC protein. (C) 2017 Elsevier Inc. All rights reserved.
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